In a continuation of studies on the dynamics of phosphate metabolism we have been investigating the properties, activities, and isozyme composition of inorganic pyrophosphatase (EC 3.6.1.1) in normal and neoplastic tissues of humans and various animals. The major enzyme from rat liver and a rat hepatoma has been purified to homogeneity, and the human placental enzyme is now being purified and characterized. Four isozymes have been identified by agarose gel electrophoresis; three in the cytosol and one (possibly two) in the mitochondria. By sequential ammonium sulfate precipitation at 50-70% saturation, and KC1 gradient and pH gradient elution from DEAE-cellulose, inorganic pyrophosphatase of human placenta has been purified to near homogeneity, exhibiting 3 bands on enzyme staining and a single diffuse band on staining with Coomassie Blue after agarose gel electrophoresis. Gel filtration yields a molecular weight of about 45,000. All 3 forms have an absolute requirement for Mg[unreadable]2+[unreadable], a Ks for PPi of 8-12 micromolar and Ka for Mg[unreadable]2+[unreadable] of approximately 1 mM at a PPi concentration of 0.2 mM. The pH optimum is 7.2. It is inhibited by Ca[unreadable]2+[unreadable], (Ki = 0.2 mM); Mn[unreadable]2+[unreadable], (Ki = 0.4 mM); Co[unreadable]2+[unreadable], (Ki = 1 mM); F, (Ki = 0.25 mM); Zn[unreadable]2+[unreadable], Ki = 5 mM. It is not inhibited by p-chloromercuribenzoate, bromoacetate or iodoacetate at 10 mM. Hg[unreadable]2+[unreadable] inhibits about 40% at 0.01 mM, but inhibits about 90% on standing at 25~ for 10 min at 0.01 mM. The purified preparation has no alkaline phosphatase activity when measured with p-nitrophenyl phosphate as substrate at pH 10.4. (B)